Journal: Mucosal immunology

Article Title: Notch ligand Delta-like 4 induces epigenetic regulation of Treg cell differentiation and function in viral infection

doi: 10.1038/s41385-018-0052-1

Figure Lengend Snippet: DLL4-enriched permissive histone mark H3K4me3 around Foxp3 promoter and consensus non-coding sequences during iTreg differentiation. a 2 × 10 6 of naive CD4 T cells were skewed toward Th0 or iT reg differentiation with or without DLL4 stimulation in vitro. Chromatin immunoprecipitation were performed to detect H3K4me3 around Foxp3 promoter, consensus non-coding sequence (CNS)1, CNS2, CNS3 after 72 h. b Changes of H3K4me and H3K4me3 by DLL4 stimulation during iT reg differentiation was detected at 48 h of skewing. c H3K4me3 kinetics at Foxp3 CNS1 after 6 h, 24 h, 48 h and 72 h post iT reg differentiation were measured with or without DLL4 stimulation in vitro. Data represent mean ± SEM. Data were from one experiment representative of two to three experiments. * P < 0.05; ** P < 0.005; *** P < 0.0005; NS, no significance (unpaired two-tailed t -test)

Article Snippet: Naive T cells (10 6 /0.5 mL) were stimulated with plate-bound anti-CD3 (2.5 μg/mL; eBioscience), soluble anti-CD28 (3 μg/mL; eBioscience), and plate-bound recombinant DLL4 (1.65 μg/mL, R&D).

Techniques: In Vitro, Chromatin Immunoprecipitation, Sequencing, Two Tailed Test

Journal: Mucosal immunology

Article Title: Notch ligand Delta-like 4 induces epigenetic regulation of Treg cell differentiation and function in viral infection

doi: 10.1038/s41385-018-0052-1

Figure Lengend Snippet: Smyd3 regulated DLL4-enhanced iT reg differentiation and function in vitro. a Naive CD4 T cells (10 6 ) from wild-type B6 spleen were activated (Th0) or differentiated to Th1, Th2, Th17, Th9, iT reg , and IL-27 T R 1. After 72 h, SMYD3 were blotted. b Naive CD4 T cells (10 6 ) from Cre− control or Cre + CD4-specific SMYD3 conditional knockout (cKO) were differentiated to iT reg . After 72 h, SMYD3 level were quantified by western blotting. c Smyd3 mRNA level were measure in iT reg and DLL4-stimulated iT reg differentiation at 24, 48, and 72 h from Cre− control and Cre + Smyd3 cKO. d Naive CD4 T cells (2 × 10 6 ) from Cre− control with or without DLL4 stimulation or Cre + CD4-specific SMYD3 were differentiated to iT reg . After 72 h, H3K4me3 were precipitated, and Foxp3 promoter and CNS1, 2, 3 were qPCR quantified compared with input control. e Naive CD4 T cells (10 6 ) from Cre− control or Cre + SMYD3 cKO were activated (Th0) or differentiated to iT reg with or without DLL4. After 72 h of differentiation, Foxp3 and CD25 were labeled and quantified by flow cytometry. f Naive CD4 T cells (10 6 ) from Cre− control or Cre + SMYD3 cKO were activated (Th0) or differentiated to iT reg with or without DLL4. After 72 h of differentiation, both Th0 and iT reg were rested in IL-2 10 ng/mL for another 72 h. Foxp3 were detected and quantified by flow cytometry. g After 6 days of iT reg differentiation with DLL4 as described in f , viable DLL4-exposed iT reg were sorted out as DAPI − CD25+ and co-cultured with Cell Trace violet (CTV) labeled CD45.1+ naive T cells with anti-CD3/anti-CD28 beads. After 3 days in co-culture, proliferation was assessed by CTV dilution in CD45.1 + responder cells. Data represent mean ± SEM. Data were from one experiment representative of two to three experiments. * P < 0.05; ** P < 0.005; *** P < 0.0005; NS, no significance (unpaired two-tailed t -test)

Article Snippet: Naive T cells (10 6 /0.5 mL) were stimulated with plate-bound anti-CD3 (2.5 μg/mL; eBioscience), soluble anti-CD28 (3 μg/mL; eBioscience), and plate-bound recombinant DLL4 (1.65 μg/mL, R&D).

Techniques: In Vitro, Control, Knock-Out, Western Blot, Labeling, Flow Cytometry, Cell Culture, Co-Culture Assay, Two Tailed Test

Journal: Mucosal immunology

Article Title: Notch ligand Delta-like 4 induces epigenetic regulation of Treg cell differentiation and function in viral infection

doi: 10.1038/s41385-018-0052-1

Figure Lengend Snippet: DLL4/Notch upregulated SET and MYND domain containing protein 3 ( Smyd3 ). a The expression of 87 epigenetic enzymes in epigenetic PCR array (SA Bioscience, PAMM-085) were measured in DLL4-stimulated iTreg compared to Th0 after 48 h of skewing. Fold changes were indicated with a heatmap. The following genes were either up or downregulated more than twofold: Dnmt1 (A11), Hdac9 (C7), Nek6 (E2), Smyd3 (G4), Ube2a (G7), Usp21 (G11). b All the methyltransferases in the PCR array were presented. The arrow indicated Smyd3 as the most-expressed methyltransferase in vitro. c Smyd3 expression level were measured after 24 h of Th0 and iT reg differentiation with or without DLL4 activation in vitro. d Kinetics of Smyd3 expression during Th0 and iT reg differentiation with or without DLL4 activation at 24, 48, 72 h. e Naive CD4 T cells were isolated from wild-type B6 or CD4-specific Dominant-negative MAML1 (DNMAML) mice. Smyd3 were measured after 48 h of iT reg differentiation + DLL4 stimulation in vitro. f Naive CD4 T cells were isolated from CD4-specific Rbpj knockout mice (Rbpj cKO) or Wild-type unfloxed littermate control (wt). Activated (Th0) or iT reg differentiation with or without DLL4 were performed. After 48 h, CD4 T cells were harvest and Smyd3 were measured. g SMYD3 expression was blotted after 72 h of iT reg differentiation in wt and Rbpj cKO. The relative SMYD3 level in Rbpj cKO versus wt were quantified by densitometry software (ImageJ) and normalized with internal control—β-actin. This was repeated in 3 different experiments with similar results. h Naive CD4 T cells (2X10 6 ) were isolated from wild-type B6 mice. After iT reg differentiation with or without DLL4 stimulation for 6 h, RBP-Jκ were precipitated and RBP-Jκ-bound DNA sequence were PCR with Smyd3 primer P0 and P2. Data represent mean ± SEM. Data were from one experiment representative of two to three experiments. PCR array is one-time experiment. * P < 0.05; ** P < 0.005; *** P < 0.0005; NS, no significance (unpaired two-tailed t -test)

Article Snippet: Naive T cells (10 6 /0.5 mL) were stimulated with plate-bound anti-CD3 (2.5 μg/mL; eBioscience), soluble anti-CD28 (3 μg/mL; eBioscience), and plate-bound recombinant DLL4 (1.65 μg/mL, R&D).

Techniques: Expressing, In Vitro, Activation Assay, Isolation, Dominant Negative Mutation, Knock-Out, Control, Software, Sequencing, Two Tailed Test

Journal: Mucosal immunology

Article Title: Notch ligand Delta-like 4 induces epigenetic regulation of Treg cell differentiation and function in viral infection

doi: 10.1038/s41385-018-0052-1

Figure Lengend Snippet: DLL4 inhibition changed Th cytokine profile in vivo during RSV infection. a Wild-type Balb/c mice were intratracheally infected with Line 19 RSV. DLL4 expression were measured at 6 days post infection (dpi). N = 4 in each group. b DLL4 were neutralized at 0, 2, 4, 6 dpi. Periodic acid-Schiff (PAS) staining of formalin-fixed lung section from 8 dpi. Bar, 200 μm. ↑ indicates the detection of mucin, and designates mononuclear cells aggregates. c Smyd3 expression in mediastinal lymph node (mLN) were measured at 6 dpi after DLL4 inhibition. N = 10 in each group. d Foxp3-EGFP knock-in mice were intratracheally infected with RSV. DLL4 were neutralized at 0, 2, 4 dpi. mLN cells were collected. Viable CD4 + Foxp3-EGFP+/− cells were sorted from mLN and Smyd3 expression were measured. N = 3 in each group. e Viable CD4 + Foxp3-EGFP+/− cells were sorted from mLN. Foxp3 mRNA were measured. N = 3 in each group. f Il5 mRNA in both Foxp3-EGFP− and Foxp3-EGFP+ were measured. g Ifng mRNA in both Foxp3-EGFP− and Foxp3-EGFP+ were measured. h Foxp3-EGFP knock-in mice were intratracheally infected with RSV. DLL4 were neutralized at 0, 2, 4, 6 dpi. mLN cells were collected at 8 dpi. Viable CD4 + Foxp3-EGFP+/− cells were sorted from mLN, and Il17a expression in both Foxp3-EGFP− and Foxp3-EGFP+ were measured. i Il10 expression in both Foxp3-EGFP− and Foxp3-EGFP+ viable CD4 T cells in mLN were measured at 8 dpi. j IL-10 production by control IgG or anti-DLL4-treated mLN cells were harvest at 8 dpi and measured after 48 h of RSV re-stimulation. Data represent mean ± SEM. Data were from one experiment representative of two experiments with 3~10 mice per time point, with samples from each mouse processed and analyzed separately. * P < 0.05; ** P < 0.005; *** P < 0.0005; NS, no significance (unpaired two-tailed t -test)

Article Snippet: Naive T cells (10 6 /0.5 mL) were stimulated with plate-bound anti-CD3 (2.5 μg/mL; eBioscience), soluble anti-CD28 (3 μg/mL; eBioscience), and plate-bound recombinant DLL4 (1.65 μg/mL, R&D).

Techniques: Inhibition, In Vivo, Infection, Expressing, Staining, Knock-In, Control, Two Tailed Test

Journal: Mucosal immunology

Article Title: Notch ligand Delta-like 4 induces epigenetic regulation of Treg cell differentiation and function in viral infection

doi: 10.1038/s41385-018-0052-1

Figure Lengend Snippet: Intracellular Notch and Smyd3 in CD4 T cells supported IL-10 production and inhibited IL-17A production in vitro. a Naive CD4 T cells were isolated and incubated with DMSO or 1 μm of gamma-secretase inhibitor (gsi) and proceed to Th0 or iTreg differentiation with or without DLL4 for 24 h. Il10 mRNA was detected. b After same procedure mentioned above, Il17a mRNA were detected. c Naive CD4 T cells from DNMAML mice and Rbpj cKO were activated with DLL4 for 48 h. IL-10 production in supernatant were measured. d Naive CD4 T cells from DNMAML mice and Rbpj cKO were differentiated to iTreg with DLL4 for 48 h. IL-17A production in supernatant were measured. e Naive CD4 T cells from Cre− control or Smyd3 cKO were differentiated to iTreg with or without DLL4 for 48 h. Il10 expression were detected. f After same procedure mentioned above, Il17a mRNA were detected. g Naive CD4 T cells from Cre-control or Cre + Smyd3 cKO were differentiated to iTreg with DLL4 stimulation for 72 h then rested in IL-2 10 ng/mL for another 72 h. Viable rested iTreg (5 × 10 5 ) were re-stimulated with Th17 skewing condition for 72 h. IL-17A in Th17 re-stimulation culture were measured. Data represent mean ± SEM. Data were from one experiment representative of two to three experiments. * P < 0.05; ** P < 0.005; *** P < 0.0005; NS, no significance (unpaired two-tailed t -test)

Article Snippet: Naive T cells (10 6 /0.5 mL) were stimulated with plate-bound anti-CD3 (2.5 μg/mL; eBioscience), soluble anti-CD28 (3 μg/mL; eBioscience), and plate-bound recombinant DLL4 (1.65 μg/mL, R&D).

Techniques: In Vitro, Isolation, Incubation, Control, Expressing, Two Tailed Test

Journal: Mucosal immunology

Article Title: Notch ligand Delta-like 4 induces epigenetic regulation of Treg cell differentiation and function in viral infection

doi: 10.1038/s41385-018-0052-1

Figure Lengend Snippet: Smyd3 deletion changed gene expression profile in the presence of DLL4 stimulation during iT reg differentiation in vitro. a Principle component analysis of RNA-seq data sets of iT reg cells from Cre− control or Cre + Smyd3 cKO with or without DLL4 stimulation, assessed using the top 500 genes with the highest variance. Each symbol represented a single mouse. Each group have three biological triplicates. b Volcano plot showed the differentially expressed genes (DEGs) in control iT reg + DLL4 over control iT reg ; red dots indicated genes that were significantly (BH-adjusted p -value < 0.05) upregulated by DLL4 more than twofold (log2FoldChange > 1) and blue dots represents genes that were significantly downregulated by DLL4 more than twofold. The y axis represented the significance of the differential analysis. c Venn diagram demonstrated the overlap between current DLL4-regulated DEGs and published T reg signatures and Foxp3-dependent genes. d Gene Ontology (GO) of un-overlapped DLL4-regulated DEGs (781 genes). Treemap showed the significant GO term with False-discovery-rate (FDR)-adjusted global P -value < 0.05. P -value inversely proportional to area for each GO term. e Normalized expression of all the DEGs that were significantly upregulated by DLL4 stimulation for more than 2.00-fold, and downregulated more than 1.51-fold by Smyd3 deletion. Heatmap presented Z -score in a dendrogram clustered by Pearson’s distribution and ranked based on rowMeans. Top 20 DEGs and Deltex1 were indicated. f Naive CD4 T cells from wild-type control or Rbpj cKO were differentiated to iT reg with or without DLL4 for 24 h. Lag3 expression were detected. g Naive CD4 T cells from Cre− control or Smyd3 cKO were differentiated to iT reg with or without DLL4 for 48 h. Lag3 expression were detected

Article Snippet: Naive T cells (10 6 /0.5 mL) were stimulated with plate-bound anti-CD3 (2.5 μg/mL; eBioscience), soluble anti-CD28 (3 μg/mL; eBioscience), and plate-bound recombinant DLL4 (1.65 μg/mL, R&D).

Techniques: Gene Expression, In Vitro, RNA Sequencing, Control, Expressing

Journal: Mucosal immunology

Article Title: Notch ligand Delta-like 4 induces epigenetic regulation of Treg cell differentiation and function in viral infection

doi: 10.1038/s41385-018-0052-1

Figure Lengend Snippet: Schematic summary of how DLL4 promotes Smyd3 expression to confer regulatory features of CD4 T cells

Article Snippet: Naive T cells (10 6 /0.5 mL) were stimulated with plate-bound anti-CD3 (2.5 μg/mL; eBioscience), soluble anti-CD28 (3 μg/mL; eBioscience), and plate-bound recombinant DLL4 (1.65 μg/mL, R&D).

Techniques: Expressing